Germacranolides and a New Type of Guaianolide from

The aerial parts of an Argentinian collection of Acanthospermum hispidum afforded 26 sesquiterpene lactones, including the two guaianolides (1 and 2) ...
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© Copyright 2000 by the American Chemical Society and the American Society of Pharmacognosy

Volume 63, Number 10

October 2000

Full Papers Germacranolides and a New Type of Guaianolide from Acanthospermum hispidum Elena Cartagena,† Alicia Bardo´n,‡ Ce´sar A. N. Catala´n,‡ Zaida N. J. de Herna´ndez,§ Luis R. Herna´ndez,§ and Pedro Joseph-Nathan*,§ Instituto de Quı´mica Orga´ nica, Facultad de Bioquı´mica, Quı´mica y Farmacia, Universidad Nacional de Tucuma´ n, Ayacucho 491, S. M. de Tucuma´ n 4000, Argentina, and Departamento de Quı´mica, Centro de Investigacio´ n y de Estudios Avanzados, Instituto Polite´ cnico Nacional, Apartado 14-740, Me´ xico, D. F. 07000, Me´ xico Received October 11, 1999

The aerial parts of an Argentinian collection of Acanthospermum hispidum afforded 26 sesquiterpene lactones, including the two guaianolides (1 and 2) having a novel oxygen bridge between C-4 and C-14, three new cis,cis-germacranolides (4, 7, and 8), and two new melampolides (25 and 26). Guaianolides 1 and 2 seem to derive biosynthetically from the germacranolide 27 having the 1D14,15D5 conformation. The structures were elucidated using extensive spectroscopic analysis. Previous investigations of Acanthospermum species1-4 have led to the isolation of cis,cis-germacranolides and melampolides,2 in agreement with the fact that many species of the genera Acanthospermum, Melampodium, and Lecocarpus, belonging to the tribe Heliantheae, subtribe Melampodiinae, contain melampolides.5 In view of the fact that these compounds show cytotoxic and in vivo anticancer activity,3 and as a continuation of our work on sesquiterpene lactones of the Argentinian species of Asteraceae,6-8 we have carried out an exhaustive examination of the minor constituents of A. hispidum (Asteraceae), a shrub indigenous to northern Argentina. The aerial parts afforded the new guaianolides hispidunolides A (1) and B (2) with an unprecedented oxygen bridge between C-4 and C-14; the new cis,cis-germacranolides 4, 7, and 8; the new melampolides 25 and 26; and the known sesquiterpene lactones 3, 5, 6, 9-18, previously isolated from American1,2 and African3,4 collections of A. hispidum; compounds 1923, previously found in species of Lecocarpus from Ecua* To whom correspondence should be addressed. Tel.: (52)5747-7112. Fax: (52)5747-7113. E-mail: [email protected]. † Universidad Nacional de Tucuma ´ n. ‡ Universidad Nacional de Tucuma ´ n, Research Member of the National Research Council of Argentina (CONICET). § Centro de Investigacio ´ n y de Estudios Avanzados.

10.1021/np9905057 CCC: $19.00

dor;5,9 compound 24, previously isolated from an Australian collection of Siegesbeckia orientalis;10 and loliolide.11 Results and Discussion Hispidunolide A (1) showed IR bands for alcohol, γ-lactone, and ester groups at 3450, 1760, and 1735 cm-1, respectively. It has the molecular formula C22H28O8 as followed from its mass spectrum, which showed a [M]+ at m/z 420, accounting for nine degrees of unsaturation. Mass spectral peaks at m/z 360 [M - CH3COOH]+, 335 [M C5H9O]+ and 85 [C5H9O]+ indicated the presence of an acetate and a saturated five-carbon atom ester. The 1H NMR spectrum showed the typical signals of a 2-methylbutyrate residue at δ 2.37 (qt, J ) 7.0, 7.0 Hz), 1.62 (ddq, J ) 13.5, 7.0, 7.0 Hz), 1.44 (ddq, J ) 13.5, 7.0, 7.0 Hz), 1.08 (d, J ) 7.0 Hz), and 0.89 (t, J ) 7.0 Hz). An R-methylene-γ-lactone moiety was evident by the two doublets at δ 6.28 (J ) 3.5 Hz) and 5.56 (J ) 3.0 Hz) in the 1H NMR spectrum. The 13C NMR spectrum showed 22 signals corresponding to three CH3, five CH2, eight CH, and six quaternary carbons, as deduced from a DEPT experiment, in agreement with the molecular formula obtained from the mass spectrum. The 13C NMR spectrum also indicated the presence of a lactone moiety, which showed signals at δ 168.8 (C-12), 134.6 (C-11), and 122.1

© 2000 American Chemical Society and American Society of Pharmacognosy Published on Web 08/11/2000

1324 Journal of Natural Products, 2000, Vol. 63, No. 10

(C-13). The guaianolide skeleton was easily deduced from the 1H NMR spectrum and sequential spin decoupling involving H-1, H-5, H-6, and H-7. The vinyl proton signal at C-14 appeared at δ 6.30 in agreement with the chemicalshift range for protons attached to the R-carbon in enolethers.12 The COSY experiment showed that the signal at δ 6.30 (H-14) was long-range coupled with the signals at δ 5.06 and 2.91, corresponding to H-9 and H-1, respectively. The signals at δ 114.2 and 140.5 were assigned to the enolether carbons C-10 and C-14, respectively. To establish the relative configuration of the fragment C-1-C-5-C-6-C-7 and that of C-4, the minimum energy conformations of 1, having either a C4β-O-C14 or a C4R-O-C14 bridge, was calculated using the PCMODEL program.13 These calculations showed that the dihedral angles and coupling constant values for the fragment CH(1)-CH(5)-CH(6)-CH(7) in the C4R-O-C14 isomer are: HβC(1)-HβC(5) ) 56° (J ) 3.6 Hz), HβC(5)-HβC(6) ) -49° (J ) 5.1 Hz), and HβC(6)-HRC(7) ) -174° (J ) 11.2 Hz); while for the C4β-OC14 isomer the values are: HRC(1)-HRC(5) ) -48° (J ) 5.0 Hz), HRC(5)-HβC(6) ) -142° (J ) 6.6 Hz), and HβC(6)-HRC(7) ) -165° (J ) 10.8 Hz). The latter set of values was in good agreement with the observed coupling constants, as can be seen in Table 1. To confirm the β-orientation of the vinyl oxygen at C-4, an NOE experiment irradiating the H-9 signal showed enhancement of the signal at δ 6.30 (6%) corresponding to H-14. The minimum energy conformation of 1 is shown in Scheme 1. The individual assignment of the protons attached to C-3 was deduced from the minimum energy conformation of hispidunolide A (1), in which the dihedral angles and calculated coupling constants are: HRC(2)-HRC(3) ) 14° (J ) 11.5 Hz), HRC(2)-HβC(3) ) -106° (J ) 1.5 Hz), HβC(2)-HRC(3) ) 134° (J ) 6.5 Hz), and HβC(2)-HβC(3) ) 13° (J ) 11.5 Hz), in good agreement with the experimental coupling

Cartagena et al.

constants given in Table 1. The small coupling constant between H-7 and H-8 indicated that the ester residue at C-8 is β-oriented. Therefore, with the small coupling constant between H-8 and H-9 also taken into account, the acetate group at C-9 is R-oriented, as occurs in many known sesquiterpene lactones isolated from this species1,2 and also found in the present investigation. The mass spectrum of hispidunolide B (2) showed [M]+ at m/z 418, corresponding to the molecular formula C22H26O8 in agreement with the 13C NMR spectrum and DEPT experiment, which showed 22 signals, three of them corresponding to CH3, four to CH2, eight to CH, and seven to quaternary carbons. Relevant mass peaks at m/z 358 [M - CH3COOH]+, 335 [M - C5H7O]+, and 83 [C5H7O]+ were indicative of an acetate and an unsaturated fivecarbon atom ester. Both the 1H and 13C NMR signals of hispidunolide B (2) indicated the presence of the same skeleton as in hispidunolide A (1), with the only difference being the ester moiety at C-8, since for 2 the 1H NMR spectrum shows signals at δ 1.96 (dq, J ) 7.0, 1.5 Hz), 1.81 (dq, J ) 1.5, 1.5 Hz), and 6.15 (qq, J ) 7.0, 1.5 Hz), which indicated the presence of an angelate residue. The 13C NMR spectrum further confirmed the angelate moiety due to the signals at δ 165.6, 141.1, 126.4, 20.5, and 16.0.14 It is interesting to note that, from the biosynthetic point of view, hispidunolides A (1) and B (2) might be biosynthesized through a hetero Diels-Alder transformation15 from germacranolide 27, which has the 1D14, 15D5 conformation,16 as shown in Scheme 1. Compound 3 was previously reported by Kraus et al.,4 but no spectral data were provided to support the structure. Therefore, the 1H and 13C NMR data of 3 are included in Tables 2 and 4, respectively. Compound 4 was isolated as a gum. The 1H NMR data indicated that we were dealing with a germacranolide-type

Germacranolides and a Guaianolide from Acanthospermum Table 1. 1Ha and

13C

Journal of Natural Products, 2000, Vol. 63, No. 10 1325

NMRb Data (CDCl3, TMS) for Hispidunolide A (1) and B (2)c 1

2

δH

1 2a 2b 3R 3β 4 5 6 7 8 9 10 11 12 13a 13b 14 15a 15b OAc

δC

2.91 br t (5.5) 1.99d 1.99d 1.77 ddd (10.5, 10.0, 3.5) 2.23 br t (10.5)

32.8 29.3

88.6 45.8 75.6 46.0 71.9 73.7 114.2 134.6 168.8e 122.1

6.28 d (3.5) 5.56 d (3.0) 6.30 br s 3.91 br s 3.91 br s 2.15 s

32.8 29.4 36.9

2.53 t (5.5) 4.48 dd (9.5, 5.5) 3.51 dddd (9.5, 3.5, 3.0, 1.5) 5.56 t (1.5) 5.15 d (1.5)

6.29 d (3.5) 5.60 d (3.0) 6.31 br s 3.96 d (17.0) 3.90 d (17.0) 2.16 s

140.5 64.3 168.9,e 21.0 b

δC

2.94 br t (5.5) 1.99d 1.99d 1.78 ddd (10.5, 10.0, 3.5) 2.23 br t (10.5)

36.9

2.52 t (5.5) 4.46 dd (9.5, 5.5) 3.48 dddd (9.5, 3.5, 3.0, 1.5) 5.48 t (1.5) 5.06 d (1.5)

a

δH

88.6 45.9 75.8 46.0 72.1 73.6 114.3 134.6 168.8e 122.3 140.6 64.3 169.0e, 21.0

c

300 MHz. J values are given in Hz in parentheses. 75.4 MHz. Other signals for 1: 2-MeBu: δH: 2.37 (qt, 7.0, 7.0, H-2′); 1.62 (ddq, 13.5, 7.0, 7.0, H-3′a); 1.44 (ddq, 13.5, 7.0, 7.0, H-3′b); 1.08 (d, 7.0, H-5′); 0.89 (t, 7.0, H-4′); δC : 174.9 (C-1′); 41.1 (C-2′); 26.6 (C-3′); 16.8 (C-5′); 11.6 (C-4′). For 2: Ang: δH: 6.15 (qq, 7.0, 1.5, H-3′); 1.96 (dq, 7.0, 1.5, H-4′); 1.81 (dq, 1.5, 1.5, H-5′); δC: 165.6 (C-1′); 141.1 (C-3′); 126.4 (C-2′); 20.5 (C-5′); 16.0 (C-4′). d Overlapping signals. eInterchangeable. Scheme 1. Proposed Biosynthetic Path and Minimum Energy Conformation of 1 and 27

Table 2. 1H NMR Data (δ, CDCl3, TMS) for Germacranolides 3, 4, 7, and 8a H-1 H-2R H-2β H-3R H-3β H-5 H-6 H-7 H-8 H-9R H-9β H-13a H-13b H-14 H-15a H-15b OAc H-2′ H-3′a H-3′b H-4′ H-5′ a

3

4

7

8

6.62 ddd (8.0, 7.0, 1.5) 2.70 mb 2.85 dddd (15.0, 7.0, 4.0, 1.0) 2.69 mb 2.40 ddd (14.5, 7.5, 4.0) 5.55 br d (9.5) 5.46 dd (9.5, 4.0) 2.64 dddd (4.0, 3.0, 2.5, 2.5) 5.93 ddd (10.0, 7.0, 2.5) 3.07 br ddd (14.0, 7.0, 1.5) 2.40 ddd (14.0, 10.0, 1.5) 6.35 d (3.0) 5.71 d (2.5) 9.41 d (1.5) 4.49 s 4.49 s 2.12 s 2.49 sept (7.0) 1.12 d (7.0)

6.63 ddd (8.5, 6.5, 1.5) 2.70 mb 2.85 dddd (15.0, 6.5, 4.0, 1.0) 2.69 mb 2.40 ddd (14.5, 7.5, 4.0) 5.57 br d (9.5) 5.48 dd (9.5, 4.0) 2.67 dddd (4.0, 3.0, 2.5, 2.5) 5.98 ddd (10.0, 7.0, 2.5) 3.07 br ddd (14.0, 7.0, 1.5) 2.57 ddd (14.0, 10.0, 1.5) 6.36 d (3.0) 5.72 d (2.5) 9.43 d (1.5) 4.53 dd (13.5, 1.5) 4.46 dd (13.5, 1.0) 2.12 s

6.67 br t (6.5) 2.70 mb 2.85 dddd (15.0, 6.5, 4.0, 1.0) 2.69 mb 2.40 ddd (14.5, 7.5, 4.0) 5.55 br d (9.5) 5.41 dd (9.5, 4.0) 2.75 dq (4.0, 3.0, 2.5, 1.5) 6.13 ddd (10.0, 7.0, 1.5) 3.07 br dd (14.0, 7.0) 2.57 ddd (14.0, 10.0, 2.0) 6.40 d (3.0) 5.79 d (2.5) 9.45 d (2.0) 4.49 s 4.49 s 2.12 s

6.78 dd (9.0, 6.0) 3.25 dddd (15.0, 8.0, 6.0, 2.0) 2.80 mb 2.58 ddd (14.0, 8.0, 2.0) 2.99 ddd (14.0, 11.0, 8.0) 5.58 br d (9.5) 5.43 dd (9.5, 4.5) 2.75 mb 6.53 dd (9.0, 2.5)

6.09 qq (7.0, 1.5)

6.15b

1.09 d (7.0)

1.96 dq (7.0, 1.5) 1.81 dq (1.5, 1.5)

1.96 dq (7.0, 1.5) 1.85 dq (1.5, 1.5)

5.80 dd (9.0, 2.0) 6.41 d (3.0) 5.86 d (2.5) 9.40 d (2.0) 4.08 s 4.08 s 2.00 s 2.31 sext (7.0) 1.60 mb 1.39 m 0.85 t (7.0) 1.05 d (7.0)

300 MHz. J values are given in Hz in parentheses. b Overlapping signals.

sesquiterpene lactone containing acetate and angelate esters. These data are similar to those of related cis,cisgermacranolides with 2-methylbutyrate or isovalerate ester

residues attached to C-8.2,4 The presence of a 1,10-cisdouble bond with an aldehyde group at C-10 followed from the chemical shifts of H-1 (δ 6.63, ddd) and H-14 (δ 9.43,

1326 Journal of Natural Products, 2000, Vol. 63, No. 10

Cartagena et al.

Table 3. 1H NMR Data (δ, CDCl3, TMS) for Melampolides 10, 14, 16, 25, and 26a,b 10

14

16

25

26

H-1 H-2a H-2b H-3R H-3β H-5 H-6 H-7

6.63 ddd (9.5, 7.0, 2.0) 2.47c 2.42c 2.04 ddd (12.5, 12.5, 2.0) 2.83 ddd (12.5, 6.0, 2.5) 5.16 br d (10.5) 5.23 t (10.5) 2.48c

6.82 dd (10.0, 7.5) 2.74 m 2.62c 2.04 ddd (12.0, 12.0, 2.0) 2.85 ddd (12.0, 5.5, 2.5) 5.03 br d (10.0) 5.20 t (10.0) 2.63c

H-8 H-9R H-9β H-13a H-13b H-14a H-14b H-15a H-15b

6.36 ddd (10.0, 8.0, 2.0) 2.75 ddd (14.0, 8.0, 2.0) 2.06 ddd (14.0, 10.0, 1.5) 6.24 d (3.5) 5.58 d (3.0) 9.46 d (1.5)

6.65 dd (8.5, 1.5)

5.80 dd (9.0, 8.0) 2.42 mc 2.34 mc 1.90 ddd(12.5, 12.5, 2.5) 2.69 ddd (12.5, 5.5, 2.5) 5.15 br d (10.0) 5.35 t (10.0) 3.35 dddd (10.0, 3.5, 3.0, 2.0) 6.16 dd (9.5, 2.0)

5.77 dd (9, 7.5) 2.44 mc 2.3c 2.0c 2.3c 5.03 br d (10.5) 5.10 dd (10.5, 9) 3.31dddd (9.0, 3.5, 3.0, 2.0) 6.16 dd (9.5, 2.0)

5.75 br dd (8.5, 7.5) 2.44 mc 2.3c 2.0c 2.3c 5.01 br d (10.5) 5.10 dd (10.5, 9) 3.29 dddd (9.0, 3.5, 3.0, 2.0) 6.06 dd (9.5, 2.0)

3.88 dd (8.5, 2.0) 6.30 d (3.5) 5.87 d (3.0) 9.52 d (2.0)

4.53 d (12.5) 4.32 br d (12.5)

4.47 br d (13.0) 4.37 br d (13.0)

5.53 d (9.5) 6.24 d (3.5) 5.68 d (3.0) 4.40 br d (12.5) 4.23 br d (12.5) 4.49 br s 4.49 br s

5.42 d (9.5) 6.24 d (3.5) 5.68 d (3.0) 4.38 d (12.5) 4.21 br d (12.5) 1.97 br s

5.35 d (9.5) 6.24 d (3.5) 5.63 d (3.0) 4.37 br d (12.5) 4.19 d (12.5) 1.98 br s

a 300 MHz. J values are given in Hz in parentheses. b Other signals (δ), for 10: i-Bu: 2.53 (sept, 7.0, H-2′); 1.14 (d, 7.0, H-4′); 1.12 (d, 7.0, H-3′a). For 14: Ang: 6.05 (qq, 7.0, 1.5, H-3′); 1.96 (dq, 7.0, 1.5, H-4′); 1.88 (dq, 1.5, 1.5, H-5′); OMe: 3.10 s. For 16: Ang: 6.11 (qq, 7.0, 1.5, H-3′); 1.95 (dq, 7.0, 1.5, H-4′); 1.82 (dq, 1.5, 1.5, H-5′); Ac: 1.95 s. For 25: Ang: 6.10 (qq, 6.0, 1.5, H-3′); 1.95 (overlapped, H-4′); 1.82 (dq, 1.5, 1.5, H-5′); Ac: 1.94 s. For 26: 2-MeBu: 2.30 (m, H-2′); 1.60 (m, H-3′a); 1.39 (m, H-3′b); 1.06 (d, 7.0, H-5′); 0.86 (t, 7.5, H-4′). Ac: 1.97 s. cOverlapping signals.

Table 4. 13C NMR Data (δ, CDCl3 , TMS) for Compounds 3, 8, 13, 16, 17, 24, and 26a 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 OAc 1′ 2′ 3′ 4′ 5′

153.2 25.0 26.1 135.0d 129.2 73.2 46.8 71.9 28.6 142.0 134.4d 169.3 124.7 194.8 66.6 170.4 20.9 175.7 34.0 19.1 18.6

8 158.9 24.9 27.3c 139.3c 127.0 73.5 46.4 72.2 69.6 140.3c 133.4 169.0 126.2 193.3 65.8 170.4 20.7 175.0 41.3 26.5c 11.6 17.1

13b

16

17

24

26

153.8 27.0 32.6 140.4d 128.4 73.8 49.4 65.6 28.8 142.7d 134.8 169.3 121.2 195.5 60.5

134.5d

134.5d

26.3 33.1 139.9 127.9 72.3 50.9 68.8 74.2 136.0 134.1d 169.4 121.3 64.0 60.9 170.0 20.8 166.5 142.0 126.8 15.8 20.4

26.5e 33.1 141.8 128.0 72.3 51.0 68.8 74.0 136.0 134.0d 169.3 121.3 64.0 60.9 169.9 21.0 175.4 41.4 26.3e 11.6 16.9

158.5 27.6 32.4 140.9d 128.5 73.4 51.1 69.8 67.9 141.2d 133.7 169.0 122.3 193.8 60.6 170.5 20.8 175.4 34.1 19.0 19.0

134.3d 26.5 37.7 139.1 125.9 72.8 50.8 68.9 75.6 136.2 134.8d 164.6 121.1 64.0 16.7

171.7 43.3 25.8 22.4 22.3

175.4 41.4 25.4 11.7 16.7

a 75.4 MHz. b Distinction of C-2 and C-3′ followed from APT measurements. c Distinction of C-4 from C-10 and of C-3 from C-3′ followed from HMBC measurements. d,e Interchangeable signals.

d).1 The cis-configuration of the 4,5-double bond was deduced from the typical chemical shifts of H-5 (δ 5.57, br d) and H-6 (δ 5.48, dd) and the value of J6,7 ) 4.0 Hz.2,5 The angelate residue at C-8 is β-oriented because J7,8 ) 2.5 Hz, in agreement with the well-known syn-periplanar orientation of H-8 and H-7.5 In addition, β-oriented ester residues at this position are frequent in germacranolides of the subtribe Melampodiinae. The large coupling constant between H-8 and H-9β (10.0 Hz) showed that H-9β is trans to H-8. This stereochemistry places H-9β and H-14 into a W relationship if the aldehyde carbonyl is oriented such that there is maximal overlap between the π orbitals of the 1(10)-double bond and the carbonyl group, an arrangement that accounts for the observed long-range coupling between H-9β and H-14.1 An allylic coupling between H-1 and H-9R was also observed. The 1H NMR spectrum of 7 (Table 2) was very similar to that of 4. It only differed in the H-8 and H-15 chemical

shifts, and therefore in 7 the angelate ester is located at C-15, while the acetate group is located at C-8. The spectral features of 8 were similar to those described for cis,cis-germacranolides 4 and 7, but no signals at δ 2.57 and 3.07 were found for the H-9 protons. Instead, a double doublet at δ 5.80 (J ) 9.0, 2.0 Hz) and a singlet at δ 2.00 revealed the presence of an R-oriented acetate at C-9, as further supported by the signals at δ 69.6 (C-9), 170.4, and 20.7 (OAc) in the 13C NMR spectrum. A singlet at δ 4.08 (2H) indicated that a hydroxyl group was bonded to a methylene group, and it was assigned to the protons at C-15. The 500-MHz HMBC contour plot showed, among others, correlations between C-5 and H-3, H-6, and H-6; between C-6 and H-5 and H-8; between C-7 and H-6, H-8, and the two hydrogens at C-13, between C-8, and H-6 and H-9, between C-9 and H-1, H-8, and H-14; between the acetate carbonyl and H-9, and between the 2-methylbutyrate carbonyl and H-8, the two H-3′ signals, and the H-5′ methyl, which further supported the structure of 8. The experiment also allowed distinction of the C-4 and C-10 signals; the former had correlations with one H-2 and one H-3, H-5, and H-15, while the latter had correlations with one H-2, H-9, and H-14. There was also distinction of the C-3 and C-3′ signals, was much as the former had correlations with H-5 and H-15, while the latter correlated with H-2′, H-4′, and H-5′. The IR spectrum of 10 showed strong absorptions at 3450, 1760, 1735, and 1685 cm-1, indicating the presence of a hydroxyl, γ-lactone, ester, and conjugated carbonyl with a double bond, respectively. The 1H NMR spectrum exhibited signals ascribable to a germacranolide-type compound bearing an isobutyrate ester, because it was similar to the spectra of related melampolides.1,2,5,9 The presence of a 1,10-cis-double bond with an aldehyde group at C-10 followed from the chemical shifts of H-1 (δ 6.63, ddd) and H-14 (δ 9.46, d).1 The trans-configuration of the 4,5-double bond was deduced from the typical chemical shifts of H-5 (δ 5.16, br d) and H-6 (δ 5.23, t), as well as a large J6,7 of 10.5 Hz.2 The signals at δ 4.53 (d) and 4.32 (br d) were assigned to H-15a and H-15b, respectively. The signal at δ 6.36 (ddd) is typical for a proton attached to a carbon supporting an ester group and was assigned to H-8. The small coupling constant between H-7 and H-8 (2.0 Hz) indicated that the ester residue at C-8 is β-oriented. An

Germacranolides and a Guaianolide from Acanthospermum

allylic coupling between H-1 and H-9R (J ) 2.0 Hz) and a W-type coupling between H-9β and H-14 (J ) 1.5 Hz) were observed. The typical signals for an R-methylene-γ-lactone moiety appeared at δ 6.24 (d, J ) 3.5 Hz, H-13a) and 5.58 (d, J ) 3.0 Hz, H-13b). A previous report on this compound by Kraus et al.4 provided no spectral data to support the structure. The 1H NMR data are given in Table 3. Compounds 25 and 26 were melampolides having an acetate and an angelate in the case of 25, and an acetate and a 2-methylbutyrate in the case of 26. They differed in the ester group attached to C-8, as can be seen from the 1H NMR data given in Table 3. Neither the IR nor NMR spectra showed bands or signals for an aldehyde group. However, in the 1H NMR spectrum of 25 an AB system at δ 4.38 (d, J ) 12.5 Hz) and 4.21 (br d, J ) 12.5 Hz), assigned to the H-14 protons, was present. The chemical shift of these protons and those of H-1 (δ 5.77, dd) were similar to those observed for related melampolides bearing a CH2OH at C-10.1 Similar signals were observed for 26, as can be seen in Table 3. Noteworthy for melampolides with carbonyl groups attached to C-10 is the chemical shift of H-1, found 1 ppm downfield. The total signal assignment was achieved by comparison with a melampolide obtained by Herz and Kalyanaraman1 by reduction of a precursor with a carbonyl attached to C-10. The 1H NMR spectrum of melampolide 14 differed from that of lecocarpinolide J (21), previously isolated from Lecocarpus lecocarpoides,5 only in the signals corresponding to the ester residue at C-8. Because 14 has an allylic methoxyl group and we used methanol for the HPLC separation, one could suspect it to be an artifact. However, lecocarpinolide J and lecocarpinolide M, which also contain an allylic methoxyl group at C-9, were found in the genus Lecocarpus,5 belonging to the same subtribe of Acanthospermum, for which no methanol was used during the isolation procedures. Compound 16 displayed similar spectral data to those of 25. However, the 1H and 13C NMR spectra of 16 accounted for an additional CH2OH group at C-4. Compounds 14 and 16 were previously reported by Kraus et al.,4 but no spectral data were provided to support the structures. Herein we report the 1H NMR data of 14 and 16 in Table 3 and the 13C NMR data of 132, 16,4 17,1 and 24,10 which were not published previously, in Table 4. Experimental Section General Experimental Procedures. IR spectra were recorded on a Perkin-Elmer 16F PC FT-IR spectrophotometer. Optical rotations were performed on a Perkin-Elmer 241 polarimeter. NMR spectra were recorded on Varian XL-300GS or Unity-500 spectrometers. The EIMS were obtained on a Hewlett-Packard 5989-A spectrometer at 20 eV. For separation of mixtures, a Gilson HPLC instrument with a 305 pump and a 112-differential refractometer detector was used. Columns: A (Beckman ultrasphere C18, 10 × 250 mm) and B (Beckman ultrasphere C8, 10 × 250 mm) were employed. Retention times (tR) were measured from the solvent peak. For column chromatography, Si gel Merck 70-230 or 230-400 mesh ASTM were used. Plant Material. The aerial parts (flowers and leaves) of A. hispidum DC. were collected in Vipos, Tucuma´n Province, Argentina, in April 1995. A voucher specimen (LIL 604458) is on deposit at the Herbarium of Fundacio´n Miguel Lillo, Tucuma´n, Argentina. Extraction and Isolation. The plant material (330 g) was extracted with CHCl3 (2 × 3 L) at room temperature for 14 days to give 15.9 g (yield 4.8%) of a crude extract, which was suspended in EtOH (130 mL) at 55 °C, diluted with H2O (100 mL), and extracted successively with hexane (3 × 150 mL) and CHCl3 (3 × 150 mL). The chloroform extract on evaporation

Journal of Natural Products, 2000, Vol. 63, No. 10 1327

at reduced pressure furnished a residue (3.38 g), which was column chromatographed over Si gel using CHCl3 with increasing amounts of EtOAc (0-100%) and finally MeOH, to give nine fractions. Fractions containing sesquiterpene lactones, as evidenced by IR, were further processed. A portion (200 mg) of fraction 2 (463 mg) was chromatographed by HPLC (Column A, MeOH-H2O, 3:2, 1.5 mL min-1) to give 5 mg of 3, tR 27 min, 4 mg of 1, tR 32 min; 2.3 mg of 4, tR 43 min; 33.3 mg of 6, tR 52 min; 2 mg of 9, tR 84 min; and mixtures further purified by HPLC (Column B, MeOH-H2O, 3:2, 1.3 mL min-1) to give 1.5 mg of 2, tR 30 min, and 0.9 mg of 5, tR 45 min. Fraction 3 (168 mg) was chromatographed by HPLC (Column A, MeOH-H2O, 4:3, 1.5 mL min-1) to give 2.1 mg of loliolide,11 tR 6 min; 5.5 mg of 2, tR 23 min; 2.4 mg of 1, tR 25 min; 1.3 mg of 25, tR 76 min; 6.4 mg of 26, tR 88 min; and mixtures further purified by HPLC (Column B, MeOH-H2O, 1:1, 1.3 mL min-1) to give 1 mg of 10, tR 24 min; 0.7 mg of 19, tR 35 min; 7.4 mg of 11, tR 28 min; 4.4 mg of 20, tR 40 min; 8.7 mg of 12, tR 44 min; and 11.4 mg of 13, tR 48 min. A portion (110 mg) of fraction 4 (193 mg) was processed by HPLC (Column A, MeOH-H2O, 1:1, 2 mL min-1) to give 1.7 mg of 24, tR 14 min; 0.6 mg of 7, tR 19 min; 5.8 mg of 13, tR 31 min; and mixtures further purified by HPLC (Column B, MeOH-H2O, 1:1, 2 mL min-1) to give 1 mg of 14, tR 12 min; 1.9 mg of 10, tR 14 min; 3.3 mg of 21, tR 16 min; 9.6 mg of 19, tR 20 min; 30.8 mg of 11, tR 25 min; and 1.6 mg of 12, tR 16 min. A portion (200 mg) of fraction 5 (423 mg) was processed by HPLC (Column B, MeOH-H2O, 6:5, 2 mL min-1) to give 96 mg of 11, tR 45 min; 4.5 mg of a mixture of 13 and 24, tR 54 min; and mixtures further purified by HPLC (Column A, MeOH-H2O, 1:1, 2 mL min-1) to give 0.5 mg of 22, tR 13 min; 3.1 mg of 24, tR 19 min; and 4.7 mg, of 19, tR 30 min. A portion (200 mg) of fraction 6 (487 mg) was chromatographed by HPLC (Column A, MeOH-H2O, 1:1, 1.8 mL min-1) to give 10.3 mg of 24, tR 15 min, and 35.8 mg of 11, tR 28 min. A portion (200 mg) of fraction 7 (549 mg) was processed by HPLC (Column A, MeOH-H2O, 1:1, 2 mL min-1) to give 1.1 mg of 15, tR 18 min; 18.8 mg of 17, tR 30 min; 8.5 mg of 8, tR 61 min; and mixtures further purified by HPLC (Column B, MeOH-H2O, 1:1, 2 mL min-1) to give 1.5 mg of 15, tR 21 min; 4.7 mg of 16, tR 27 min; and 0.7 mg of 17, tR 32 min. Fraction 8 (60 mg) was chromatographed by HPLC (Column B, MeOH-H2O, 1:1, 2 mL min-1) to give 2.3 mg of 23, tR 9 min, and 2.8 mg of 18, tR 10 min. 9-Acetyloxy-15-hydroxy-8-(2-methylbutanoyloxy)10(14),11(13)-guaiadien-6,12-olide-4,14-oxide (hispidunolide A) (1): gum; [R]25589 -46°, [R]25578 -48°, [R]25546 -56°, [R]25436 -102°, [R]25365 -174° (c 5.0, CHCl3); UV (EtOH) λmax (log ) 210 (4.1) nm; IR (CHCl3) νmax 3450, 1760, 1735, 1635 cm-1; 1H and 13C NMR, see Table 1; EIMS (direct inlet) m/z 420 [M]+ (3), 402 (9) [M - H2O]+, 360 [M - CH3COOH]+ (1), 335 [M - CH3CH2CH(CH3)CO]+ (11), 318 [M - CH3CH2CH(CH3)COOH]+ (1), 317 [M - H2O - CH3CH2CH(CH3)CO]+ (4), 293 [M - CH2CO - CH3CH2CH(CH3)CO]+ (66), 275 [M - CH3COOH - CH3CH2CH(CH3)CO]+ (74), 258 [M - CH3COOH CH3CH2CH(CH3)COOH]+ (16), 240 [M - H2O - CH3COOH - CH3CH2CH(CH3)COOH]+ (34), 85 [CH3CH2CH(CH3)CO]+ (39), 57 [C4H9]+ (100). 9-Acetyloxy-15-hydroxy-8-angeloyloxy-10(14),11(13)guaiadien-6,12-olide-4,14-oxide (hispidunolide B) (2): gum; [R]25589 -40°, [R]25578 -42°, [R]25546 -50°, [R]25436 -87°, [R]25365 -147° (c 6.2, CHCl3); UV (EtOH) λmax (log ) 209 (4.5) nm; IR (CHCl3) νmax 3500, 1760, 1730, 1640 cm-1; 1H and 13C NMR, see Table 1; EIMS (direct inlet) m/z 418 [M]+ (1), 400 [M - H2O]+ (4), 358 [M - CH3COOH]+ (0.2), 335 [M - CH3CHC(CH3)CO]+ (12), 317 (2), 293 [M - CH2CO - CH3CHC(CH3)CO]+ (35), 275 [M - CH3COOH - CH3CHC(CH3)CO]+ (41), 258 [M - CH3COOH - CH3CHC(CH3)COOH]+ (5), 240 [M - CH3COOH - CH3CHC(CH3)COOH - H2O]+ (12), 83 [CH3CHC(CH3)CO]+ (100), 55 [C4H7]+ (28). 15-Acetyloxy-8β-angeloyloxy-14-oxo-(4Z)-acanthospermolide (4): gum; UV (EtOH) λmax (log ) 211 (5.3) nm; IR (CHCl3) νmax 2720, 1760, 1735, 1630 cm-1; 1H NMR, see Table

1328 Journal of Natural Products, 2000, Vol. 63, No. 10

2; EIMS (direct inlet) m/z 402 [M]+ (1), 342 (4), 302 (15), 242 (20), 213 (11), 82 (44), 54 (100). 8β-Acetyloxy-15-angeloyloxy-14-oxo-(4Z)-acanthospermolide (7): gum; UV (EtOH) λmax (log ) 212 (5.1) nm; IR (CHCl3) νmax 2720, 1760, 1735, 1630 cm-1; 1H NMR, see Table 2; EIMS (direct inlet) m/z 402 [M]+ (2), 342 (4), 302 (10), 242 (19), 213 (11), 82 (50), 54 (100). 9r-Acetyloxy-8β-(2-methylbutanoyloxy)-14-oxo-(4Z)acanthospermolide (8): gum; [R]25589 -73°, [R]25578 -78°, [R]25546 -90°, [R]25436 -168°, [R]25365 -278° (c 6.3, CHCl3); UV (EtOH) λmax (log ) 200 (3.6) nm; IR (CHCl3) νmax 2725, 1765, 1730, 1640 cm-1; 1H and 13C NMR, see Table 2 and Table 4, respectively; EIMS (direct inlet) m/z 420 [M]+ (1), 402 (15), 342 (20), 240 (26), 212 (10), 85 (32), 57 (100). 9r-Acetyloxy-8β-angeloyloxy-14-hydroxyacanthospermolide (25): gum; UV (EtOH) λmax (log ) 204 (4.1) nm; IR (CHCl3) νmax 3455, 1760, 1735, 1630 cm-1; 1H NMR, see Table 3; EIMS (direct inlet) m/z 404 [M]+ (0.2), 386 (9), 326 (15), 226 (22), 83 (100). 9r-Acetyloxy-14-hydroxy-8β-(2-methylbutanoyloxy)acanthospermolide (26): gum; [R]25589 -20°, [R]25578 -22°, [R]25546 -24°, [R]25436 -38°, [R]25365 -67° (c 4.5, CHCl3); UV (EtOH) λmax (log ) 207 (3.8) nm; IR (CHCl3) νmax 3450, 1760, 1735, 1635 cm-1; 1H and 13C NMR, see Table 3 and Table 4, respectively; EIMS (direct inlet) m/z 406 [M]+ (1), 388 (9), 328 (17), 226 (11), 85 (32), 57 (100), 43 (56). Acknowledgment. Financial support from CoNaCyT (Me´xico) and CYTED (Spain) is acknowledged. Work in Tucuma´n was supported by grants from Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica (ANPCyT), Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas de Argentina (CONICET) and Consejo de Investigaciones de la Universidad Nacional de Tucuma´n (CIUNT). We thank M. C. Marı´a Isabel

Cartagena et al.

Cha´vez (Instituto de Quı´mica, University of Mexico) for the HMBC measurements. L.R.H. thanks Ministerio de Cultura y Educacio´n de la Nacio´n Argentina (Direccio´n Nacional de Cooperacio´n e Integracio´n Educativa Internacional) for a postdoctoral fellowship. References and Notes (1) Herz, W.; Kalyanaraman, P. S. J. Org. Chem. 1975, 40, 3486-3491. (2) Bohlmann, F.; Jakupovic, J.; Zdero, C.; King, R. M.; Robinson, H. Phytochemistry 1979, 18, 625-630. (3) Jakupovic, J.; Baruah, R. N.; Bohlmann, F.; Msonthi, J. D. Planta Med. 1986, 52, 154-155. (4) Kraus, W.; Ko¨ll-Weber, M.; Maile, R.; Wunder, T.; Vogler, B. Pure Appl. Chem. 1994, 66, 2347-2352. (5) Macı´as, F. A.; Molinillo, J. M. G.; Fischer, N. H. Phytochemistry 1993, 32, 127-131. (6) de Herna´ndez, Z. N. J.; Herna´ndez, L. R.; Catala´n, C. A. N.; Gedris, T. E.; Herz, W. Phytochemistry 1997, 46, 721-727. (7) Kotowicz, C.; Bardo´n, A.; Catala´n, C. A. N.; Cerda-Garcı´a-Rojas, C. M.; Joseph-Nathan, P. Phytochemistry 1998, 47, 425-428. (8) de Herna´ndez, Z. N. J.; Catala´n, C. A. N.; Herna´ndez, L. R.; GuerraRamı´rez, D.; Joseph-Nathan, P. Phytochemistry 1999, 51, 79-82. (9) Macı´as, F. A.; Fischer, N. H. Phytochemistry 1992, 31, 2747-2754. (10) Zdero, C.; Bohlmann, F.; King, R. M.; Robinson, H. Phytochemistry 1991, 30, 1579-1584. (11) Hodges, R.; Porte, A. L. Tetrahedron 1964, 20, 1463-1467. (12) Pretsch, E.; Clerc, T.; Seibl, J.; Simon, W. Spectral Data for Structure Determination of Organic Compounds, 2nd ed.; Springer-Verlag: New York, 1989. (13) Burket, U.; Allinger N. L. Molecular Mechanics; ACS Monograph 177; American Chemical Society: Washington, DC, 1982. (14) Joseph-Nathan, P.; Wesener, J. R.; Gu¨nther, H. A. Org. Magn. Reson. 1984, 22, 190-191. (15) Martin, S. F.; Chen, K. X.; Eary, C. T. Org. Lett. 1999, 1, 79-81. (16) Samek, Z., Harmatha, J. Collect. Czech. Chem. Commun. 1978, 43, 2779-2799.

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